High Copy Origins

OG1190 - pSF-CMV-Puro-COOH-FXa-GST-6His; 5ug

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This plasmid is compatible with all of the other DNA sequences available on this site which are all sold in the same backbone. This flexibility allows you to create any vector you require with a variety of functions simply by compiling the relevant sections from our product range.

Product Name: pSF-CMV-Puro-COOH-FXa-GST-6His

Product Code: OG1190

Size (bp): 6834

Bacterial Antibiotic Selection: KanR

Origin and Compatibility: pUC high copy derived from pBR322

Bacterial Copy Number: 500-700 per cell

Promoter: Cytomegalovirus (CMV) immediate early promoter

Promoter Expression Level: This plasmid contains the mammalian CMV promoter to drive gene expression. We have tested all of our mammalian promoters in a range of cell types and CMV is consistently the strongest in those we have studied. However there are many reports of the CMV promoter demonstrating silencing by methylation in long-term culture. For this reason we stock a range of other promoters that are compatible with this plasmid and are available on request.

Plasmid Purpose:

This plasmid is designed to express tagged proteins in mammalian cells either by transient transfection or by creating stable cell lines. It contains a puromycin resistance expression cassette using the human Ubiquitin promoter to drive expression and allow for the selection of cells containing the plasmid.

About the Peptide Tag:

This plasmid contains a c-terminal Glutathione-S-Transferase (GST) affinity tag that can be fused to a gene of interest to allow protein detection and/or purification. The sequence of the tag is:

For more information on the methods that can be used to purify proteins please see our protein tag guide.

This plasmid also contains a secondary Hexa-Histidine (6His) tag protein tag. The sequence of this tag is: HHHHHH

We provide a range of dual peptide tag plasmids. This is because some peptide tags provide specific biological properties (e.g small molecule affinity new epitopes solubility or protein secretion) that are not provided by others.

About the Cleavage Tag:

This plasmid also encodes a protease cleavage site that is designed to be positioned between your gene of interest and the tag to allow the removal of the tag following protein purification or isolation. This plasmid contains a FXa cleavage tag. The protein sequence of the cleavage tag is: IEGR?. It cleaves after the Arg residue but can also cleave less frequently at secondary basic sites. Its most common secondary cleave site is between the Gly and Arg residues although the frequency of these events is protein specific.

This plasmid has been designed to allow three types of cloning into the main MCS to join a coding sequence with the tag.

SnapFusion Cloning:

If you would like to fuse your coding sequence to the tag with minimal additional bases you can use our SnapFusion technology. This process involves amplifying your gene by PCR to add specific restriction sites onto the ends. When these sites are cut they produce an overhang that is compatible with this plasmid cut with BseRI or BsgI.

Standard Enzymes:

If you are not concerned about leaving a few extra bases between the tag coding sequence and your gene you can clone your gene into the vector using standard cloning restriction enzymes. This strategy will require you to choose which enzymes you want to use to clone your gene.

These cloning techniques use reagents sold by other companies and allow you to fuse sequences together using enzymes that chew back the DNA to leave overlapping ends/overhangs. The subsequent method of joining the DNA depends on the kit used.

Sequencing Primers:

These primers can be used to sequence a number of regions in this plasmid. There are 10 forward and 10 reverse primers that are designed to flank key restriction sites and positions in the plasmid:

Transcription Termination:This plasmid contains three alternative transcription terminators for mammalian bacterial and bacteriophage (T7) expression. This means that only the promoter needs to be changed to alter the expression system you are using. We sell multiple promoters that can be used in each of these systems. The presence of each terminator does not reduce expression in the alternative systems.

Genetic Modifications to standard parts:

CMV promoter ? an NcoI site has been ablated in the CMV promoter. This change does not reduce expression.

KanR cassette ? This KanR resistance region is not a wild type sequence it has been modified to remove all restriction sites that conflict with the SnapFast system and to retain a high level of antibiotic resistance. It has been validated in E.coli.

The Puromycin gene has been modified to remove a StuI site and an EagI site to ensure compatibility with all of our products. These are all conservative changes and do not change the amino acid sequence.

The Ubiquitin (Ub) promoter in this plasmid has been modified to remove BseRI StuI and SpeI sites. These changes do not reduce expression.